Mitochondrial respiratory electron carriers are involved in oxidative stress during heat stress in Saccharomyces cerevisiae

Abstract

In the present study we sought to determine the source of heat-induced oxidative stress. We investigated the involvement of mitochondrial respiratory electron transport in post-diauxic-phase cells under conditions of lethal heat shock. Petite cells were thermosensitive, had increased nuclear mutation frequencies, and experienced elevated levels of oxidation of an intracellular probe following exposure to a temperature of 50 degrees C. Cells with a deletion in COQ7 leading to a deficiency in coenzyme Q had a much more severe thermosensitivity phenotype for these oxidative endpoints following heat stress compared to that of petite cells. In contrast, deletion of the external NADH dehydrogenases NDE1 and NDE2, which feed electrons from NADH into the electron transport chain, abrogated the levels of heat-induced intracellular fluorescence and nuclear mutation frequency. Mitochondria isolated from COQ7-deficient cells secreted more than 30 times as much H(2)O(2) at 42 as at 30 degrees C, while mitochondria isolated from cells simultaneously deficient in NDE1 and NDE2 secreted no H(2)O(2). We conclude that heat stress causes nuclear mutations via oxidative stress originating from the respiratory electron transport chains of mitochondria.

FIG. 1

Viability of respiratory mutants after heat stress. Viability was calculated as described in Materials and Methods. Cells were grown for 48 h in YPAD before heat stress. □, JM43 [rho⁺]; ■, JM43 [rho⁻]; ◊, JM43 coq7Δ; ▵, JM43 nde1 nde2; ⧫, JM43 nde1 nde2 coq7Δ; ▴, JM43 nde1 nde2 [rho⁻]. Each point represents the mean ± SD of the results from at least three experiments.

FIG. 2

Heat-induced mutation frequency of respiratory mutants. The mutation frequency at the CAN1 locus was measured as described in Materials and Methods. Cells were grown for 48 h in YPAD before heat stress and plating onto solid agar plates containing canavanine. (A) □, JM43 [rho⁺]; ■, JM43 [rho⁻]; ▵, JM43 nde1 nde2. (B) ◊, JM43 coq7Δ; ⧫, JM43 nde1 nde2 coq7Δ; ▴, JM43 nde1 nde2 [rho⁻]. Each point represents the mean ± SD of the results from at least three experiments.

FIG. 3

Heat-induced intracellular oxidation. Intracellular oxidation of DCFH fluorescent dye was measured in cells that were grown for 48 h in YPAD and subjected to heat stress for various times as described in Materials and Methods. (A) □, JM43 [rho⁺]; ◊, JM43 coq7Δ; , JM43 coq7Δ −O₂; ⧫, JM43 nde1 nde2 coq7Δ; ⊞, JM43 [rho⁺] −O₂. (B) ■, JM43 [rho⁻]; ⊕, JM43 [rho⁻] −O₂; ▵, JM43 nde1 nde2; ▴, JM43 nde1 nde2 [rho⁻]. Each point represents the mean ± SD of the results from at least three experiments.

FIG. 4

Heat-induced hydrogen peroxide leakage from mitochondria. Hydrogen peroxide was measured in mitochondria isolated from JM43, JM43 coq7Δ, JM43 nde1 nde2, and the triple mutant JM43 nde1 nde2 coq7 at 30 and 42°C in the presence and absence of ADP as described in Materials and Methods. Each bar represents the mean ± SD of the results of four experiments.

FIG. 5

Model for electron pathways during heat exposure in post-diauxic-phase respiration. The sizes of the arrows relate to the relative electron flow. CoQ, coenzyme Q; C, cytochrome c; CO, cytochrome c oxidase; O₂, superoxide anion. (A) Electrons flow to the flavin cofactors of the dehydrogenases which are coupled to CoQ. During normal post-diauxic-phase growth, abundant ethanol is oxidized and contributes to the majority of the electron flow to CoQ. (B) If CoQ is absent from the chain, electrons become diverted to oxygen to form superoxide anion. (C) Removal of the external dehydrogenases alleviates the electron flow to oxygen.